Poster: Protein modification

Abs # 804: Site-directed mutagenesis of the PGE posttranslational modification of eEF1A

Presenter:	Ransom-Hodgkins, Wendy D., W.Ransom-Hodgkins@wmich.edu
Authors	Ransom-Hodgkins, Wendy D. (A)
Affiliations:	(A): Western Michigan University

Eukaryotic eEF1A is a multifunctional protein that can be posttranslationally modified by the attachment of phosphoglycerylethanolamine (PGE) to two glutamic acid residue side chains via an amide linkage to ethanolamine. Matrix-assisted laser desorption/ionization-mass spectrometry of carrot [¹⁴C]et-eEF1A tryptic peptides was used to identify a peptide with the PGE modification; however, this peptide had three glutamic acid residues (Ransom et al., 1998). Site-directed mutagenesis was used to determine which of the three glutamic acid residues of the peptide serves as the attachment site. An in vitro transcription/translation reaction was performed with normal carrot eEF1A cDNA and mutated carrot eEF1A (Glu₂₈₉ & Glu₃₆₄ changed to Ala) to demonstrate that both cDNAs were expressed. When [¹⁴C]ethanolamine was added to the translation reaction mixture, the wheat germ eEF1A incorporated [¹⁴C]ethanolamine as determined by autoradiography of the Western blot of the translation reaction mixture. When normal carrot cDNA was used [¹⁴C]ethanolamine incorporation increased 15% above endogenous levels. However, when mutant cDNA was added there was no increase above the wheat germ control. When we added [¹⁴C]myristate to the translation reaction mixture a [¹⁴C]-labeled peptide band comigrating with eEF1A was recovered indicating that the complete phosphatidylethanolamine modification of eEF1A was made. To identify the function of the PGE modification, mutated and normal eEF1A were expressed as recombinant proteins in E.coli. The proteins were purified and the ability for the protein to perform different reported functions of eEF1A was tested. The results of these functional assays will be reported.